Method for producing ε-poly-L-lysine

ABSTRACT

The present invention provides variant strains mass-producing εPL which are obtained by variant treatment of a strain producing εPL. For using the variant strains, the strains are cultured in a cultivated medium, εPL is mass-produced and stored in the culture solution, and the stored εPL is collected from the solution. For producing εPL, a strain which produces εPL is variant-treated, the obtained variant strain is cultivated in a culture medium to which L-lysine or L-lysine and one or more sugars are added, εPL is mass-produced and stored in the culture solution, and the stored εPL is collected from the solution. 
     The variant strains producing εPL in large quantities are preferably the strains which have tolerance to an analogue of L-lysine of Streptomyces albulus subsp. lysinopolymerus No. 346-D strain and plasmid-amplifiable variant strains of the same bacteria.

This application is a continuation of Ser. No. 07/864,182, filed Apr. 3,1992, abandoned, which is a continuation-in-part of Ser. No. 07/686,261,filed Apr. 15, 1991, and now abandoned, which is a continuation of Ser.No. 07/391,267, filed Aug. 9, 1989, and now abandoned, which is adivision of Ser. No. 07/081,405, filed Jul. 31, 1987, and now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a mutant which mass-produces ε-poly-L-lysine(abbreviated as εPL in the following), a method for using the mutant anda method for producing εPL. εPL is a high-molecular weight compound asdescribed in the following equation in which amino groups of ε-positionsof L-lysine are linked to adjacent carboxyl groups of L-lysine by amidebond formation. ##STR1##

As this material is a polymer of L-lysine which is an essentialamino-acid, it has high safety and particular physical propertiesbecause of its high cation content. Accordingly, it is useful intoiletry supplies, beauty aids, feed additives, pharmaceuticals,agricultural medicines, food additives, electronic materials, etc. isbacause of these properties.

Hitherto, this material was obtained by cultivating the strain ofStreptomyces albulus subsp. lysinopolymerus No. 346-D (deposit No. 3834of microorganisms of FRI) which is an εPL producing bacterium belongingto Streptomyces in a culture medium, separating it from the obtainedculture materials and refining it (Japanese Patent Publication59-20359).

However, εPL was produced at most 0.5g per liter of culture solutionfrom the said strain. Accordingly, the production cost of this materialwas high and its wide utilization was prevented.

The inventors of the present invention have obtained a strain whichmass-produce εPL, investigated repeatedly in order to produce εPL inlarge quantities by using the said strain, and attained the followinginvention.

SUMMARY OF THE INVENTION

The present invention provides a mutant mass-producing εPL which isobtained by mutant treatment of a strain producing εPL. Further, thepresent invention provides a method for using the mutant strain,characterized in that the mutant is cultured in a cultivated medium, εPLis mass-produced and stored in the culture solution, and the stored εPLis collected from the solution. The present invention also provides amethod for producing εPL, characterized in that a strain which producesεPL is mutation-treated, the obtained mutant is cultivated in a culturemedium to which L-lysine or L-lysine and one or more sugars are added,εPL is mass-produced and stored in the culture solution, and the storedεPL is collected from the solution. The mutant is a strain whichproduces εPL in large quantities, preferably it is a mutant which hastolerance to an analogue of L-lysine of Streptomyces albulus subsp.lysinopolymerus No.346-D strain or a chloramphenicol-treated mutantamplified of the same bacterium.

The analogue material of L-lysine is preferably either a mixture ofS-aminoethyl-L-cysteine or S-aminoethyl-L-cysteine and one or morematerials selected from the group of L-threonine, glycine, L-homoserineand L-methionine.

Further, the chloramphenicol-treated mutant of Streptomyces albulussubsp. lysinopolymerus No.346-D strain is a strain which is obtained bychloramphenicol treatment and a chloramphenicol-treated mutant 50833strain (deposit No.1110 of microorganisms of FRI) obtained bychloramphenicol treatment can be exemplified.

The following description serves to illustrate the invention morespecifically.

Firstly, a method for obtaining the mutant of the present invention isdescribed. The mutants which have tolerance to an L-lysine analogue suchas S-aminoethyl-L-cysteine are prepared, e.g., from the followingmethod.

Spores of Streptomyces albulus subsp. lysinopolymerus No.346-D strainare suspended in tris-maleic acid buffer solution (pH 9.0) andN-methyl-N-nitro-N'-nitrosoguanidine is added to the solution.

After shaking the solution, the spores are collected by centrifugation,washed with sterilized water and inoculated in a culture medium. Byshaking the medium, bacteria are cultivated. The culture mediumcontaining the bacteria (hereinafter the medium is referred to asculture solution) is diluted. Then, S-aminoethyl-L-cysteine orS-aminoethyl-L-cysteine and one or more materials selected from thegroup of glycine, L-threonine, L-homoserine and L-methionine is added toan agar culture medium having the same composition as the above medium.

In this case, S-aminoethyl-L-cysteine in concentration from 0.5 to 10 mgper ml of the agar culture medium, preferably 2 mg, orS-aminoethyl-L-cysteine in the same concentration and one or more aminoacids selected from the above group in concentration from 0.2 to 5 mgper ml of the agar culture medium, preferably 1 mg, is usable. The abovediluted culture solution is applied to this agar culture medium. Afterincubating the applied agar culture medium, S-aminoethyl-L-cysteinemutants are obtained as colonies. The mutant cultivated in the agarculture medium containing S-aminoethyl-L-cysteine alone is a tolerancemutant 81512. strain. The mutant cultivated in the agar culture mediumcontaining S-aminoethyl-L-cysteine and glycine is a tolerant mutant11011A-1 strain (deposit No.1109 of microorganisms of FRI). Further, thestrain cultivated in the agar culture medium containingS-aminoethyl-L-cysteine and L-threonine is a tolerant mutant strain81502. Fermentation Research Institute, an Agency of Industrial Scienceand Technology, Ministry of International Trade and Industry, is locatedat 1-3, Yatabemachi Higashi 1-chome, Tsukubagun, Ibarakiken, Japan 305;

Next, the chloramphenicol-treated mutant strains are obtained bychloramphenicol treatment, e.g., by the following method. Streptomycesalbulus subsp. lysinopolymerus No.346-D strain orS-aminoethyl-L-cysteine tolerance mutant strain is inoculated in aculture medium. After shaking the medium, chloramphenicol is added tothe medium, and cultivation is continued. Cultivated bacteria arecollected by centrifugation, washed, and applied to an agar culturemeduim. After static cultivation, a conventional agar culture mediumcontaining Staphylococcus aureus is overlapped on the said medium. Afterfurther cultivation, the strains which formed a large zone of inhibitionof growth of Staphylococcus aureus are the desiredchloramphenical-treated mutant 50833 strain (deposit No. 1110 ofmicroorganims of FRI). Among these mutant strains, the mycologicalproperties of the 11011A-1 strain and the 50833 strain are as follows:

1. Morphological properties:

Aerical Mycel and Substrate Mycelium of the 11011A-1 strain and the50833 strain which were grown on a sucrose-nitrate agar culture mediumat 30° C. for ten days were observed by a microscope. The result is asfollows.

1) Ramification and morphology of the sporogenesis mycelia: simpleramification, and closed spiral.

2) Spore number: several tens.

3) Surface structure and spore size: spiny, about 1.2-1.5μ, and round oroval shape.

4) Existence of flagellum spores, bacterium nucleuses and sporangia:none.

5) Insertion position of Sporophore: on Aerical Mycel.

2. Growth conditions on various culture mediums

Properties of the strains on the following various culture mediums areshown as observation results after cultivating at 30° C. for 10-14 days.

a) The 11011A-1 strain (deposit No. 1109 of microorganisms of FRI)

    ______________________________________                                        Culture     Substrate  Aerical     Soluble                                    Medium      Mycelium   Mycel       pigment                                    ______________________________________                                        Sucrose-nitrate                                                                           pale       gray        none                                       agar        yellow     brown                                                  Glucose-aspara-                                                                           pale       white,      pale                                       gine agar   yellow     powdery     yellow                                     Glycerol-aspara-                                                                          pale yellow,                                                                             bad growth  pale                                       gine agar   then,      of Aerical  yellow                                                 yellow brown                                                                             Mycel                                                  Tyrosine agar                                                                             pale       white,      brown                                                  brown      rich, powdery                                          Nutrient agar                                                                             pale       white,      none                                                   yellow     rich                                                   Yeast-maltose                                                                             pale yellow                                                                              white,      none                                       agar        wrinkled   powdery,                                                                      gray brown                                                                    spots                                                  Oatmeal agar                                                                              pale       white,      none                                                   gray       then,                                                                         gray brown                                             ______________________________________                                         b) The 50833 strain (deposit No. 1110 of micro-organisms of FRI)

    ______________________________________                                        Culture     Substrate  Aerical     Soluble                                    Medium      Mycelium   Mycel       pigment                                    ______________________________________                                        Sucrose-nitrate                                                                           pale       gray        none                                       agar        yellow     brown                                                  Glucose-aspara-                                                                           pale       white,      pale                                       gine agar   brown      powdery     yellow                                     Glycerol-aspara-                                                                          pale yellow,                                                                             bad growth  pale                                       gine agar   then,      of Aerical  yellow                                                 yellow brown                                                                             Mycel                                                  Tyrosine agar                                                                             brown      white,      brown                                                             powdery, rich                                          Nutrient agar                                                                             pale       white,      none                                                   yellow     rich                                                   Yeast-maltose                                                                             pale yellow                                                                              white,      none                                       agar        wrinkled   powdery,                                                                      gray brown                                                                    spots                                                  Oatmeal agar                                                                              pale       white,      none                                                   gray       then,                                                                         gray brown                                             ______________________________________                                    

3. Physiological properties

The physiological properties of the 11011A-1 strain and the 50833 strainare as follows.

1) Range of growth temperatures: about 15°-40° C. The optimum growthtemperature: about 30° C.

2) Liquefaction of gelatine, hydrolysis of starch and peptonization ofskim milk: all positive.

3) Coagulation of skim milk: negative.

4) Formation of melanon-like pigments: a brown pigment is produced onthe tyrosine agar culture medium.

5) Composition of cell walls: the analytical result obtained by themethod of Becker et al. (Applied Microbiology, 13, 236 (1965)) showsthat the type of diaminopimelic acid in the composition of cell wallswas L, L type.

4. Assimilability of various carbon sources on the Pridham Gottliep agarculture medium:

    ______________________________________                                                L-arabinose                                                                           -                                                                     D-xylose                                                                              -                                                                     D-glucose                                                                             +                                                                     D-fructose                                                                            +                                                                     L-rhamnose                                                                            -                                                                     D-galactose                                                                           +                                                                     sucrose -                                                                     raffinose                                                                             -                                                                     D-mannitol                                                                            +                                                                     i-inositol                                                                            +                                                                     salicin -                                                             ______________________________________                                         notes:                                                                        +: assimilate,                                                                -: dissimilate                                                           

As described above, the properties of the mutants of the presentinvention are similar to the properties of Streptomyces albulus subsp.lysinopolymerus No. 346-D strain which is an original strain. Then εPLis prepared by the present invention using the mutants obtained by theabove described methods. Further, % in this specification indicatesweight(g)/volume(ml) % unless otherwise noted.

Firstly, the obtained mutants are inoculated in a culture medium, e.g.,adding L-lysine or L-lysine and one or more sugars, and cultivated. TheεPL obtained from the culture medium containing the culture materials(abbreviated as culture solution hereinafter) is separated and purified.Any culture mediums containing carbon sources, nitrogen sources,inorganic salts and vitamin can be used. Although not limiting, a mediumcontaining 5% of glucose or 5% of glycerin as the carbon source, andammonium sulfate, L-lysine or peptone as the nitrogen source ispreferred. In the course of the cultivation, the carbon source and thenitrogen source can be added successively or continuously to the culturemedium.

When L-lysine and sugars are added to the culture solution, L-lysine andsugars can be added anytime from the beginning to the ending of thecultivation, preferably in the middle of the cultivation when pH isballing. As L-lysine to be added, L-lysine monohydrochloride can be usedin the range of 0.05-2% based on the total volume of the culturesolution, preferably 0.5%. For the sugars, one or more sugars selectedfrom the group of glucose, sucrose, maltose, starch, galactose, etc.,and glycerine can be used in the range of 0.5-5% based on the totalvolume of the culture solution, preferably 2.5% of glucose can be used.

In successive addition, L-lysine and sugars are added when the sugarconcentration in the culture solution is balls below a fixed %. Forexample, 2.5% of glucose and 0.5% of L-lysine are preferably added whenthe sugar concentration is lowered below 0.1%.

In continuous addition, a glucose solution and an L-lysine solution canbe passed through a culture tank and the culture solution can beeliminated so as to maintain, for example, the glucose concentration,e.g., at 1% and, for example, L-lysine concentration, e.g., at 0.2% inthe culture solution. Further, a defoaming agent-can be added to theculture solution.

The pH value can be left to drop up to 4.0 at the beginning of thecultivation, and then maintained at 4.0 by adding an alkali solutionsuch as an aqueous sodium hydroxide solution. After removing thecultivated bacteria with a centrifuge or a filter from the culturesolution, the filtrate is purified, decolorized and concentrated. εPL iscrystallized out of the concentrated solution by using an organicsolvent such as acetone, ethanol, etc..

The effects of the present invention are as follows.

The mutants strains of the present invention have the ability tomass-produce εPL. By cultivating the said mutants strains, the mutantsproduce more εPL than the known strains. Further, according to thepresent invention, when the mutant strains of the strains producing εPLare cultivating, the mutants can produce εPL in large quantities byadding L-lysine or L-lysine and one or more sugars to the culturesolution.

Accordingly, the production cost of εPL can be greatly lowered incomparison with the conventional methods.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following non-limiting examples illustrate the present inventionmore specifically.

EXAMPLE 1

Obtention of mutants strains tolerant to S-aminoethyl-L-cysteine:

Spores of Streptomyces albulus subsp. lysinopolymerus No. 346-D strainin quantities of a platinum earpick were suspended in tris-maleic acidbuffer solution (pH 9.0) and N-methyl-N-nitro-N'-nitrosoguanidine wasadded to the solution. After shaking the solution at 30° C. for 30minutes, the spores were collected with a centrifuge, washed withsterilized water and inoculated in 5 ml of the culture medium of pH 6.8that contains 5% of glucose, 1% of ammonium sulfate, 0.5% of yeastextract, 0.136% of potassium dihydrogen phosphate heptahydrate, 0.158%of disodium hydrogen phosphate dodecahydrate, 0.05% of magnesium sulfateheptahydrate, 0.004% of zinc sulfate heptahydrate, 0.003% of ferroussulfate heptahydrate (the medium is abbreviated as the first culturemedium hereinafter). By shaking cultivation at 30° C. for a whole dayand night, bacteria were grown.

The culture solution was diluted 5000-fold by adding the MS solutionwhich contains of 0.05% of magnesium sulfate heptahydrate, 0.5% ofsodium chloride and 0.05% of Tween 80 (Trade mark). Then, the dilutedculture solution was applied to the agar culture medium having the samecomponent as that of the first culture medium in addition toS-aminoethyl-L-cysteine or S-aminoethyl-L-cysteine in concentration of 2mg per ml of the agar culture medium and glycine or L-threonine inconcentration of 1 mg per ml of the agar culture medium. The appliedagar culture medium was incubated at 30° C. for 48 hours to grow strainsas colonies, and mutants strains tolerant to S-aminoethyl-L-cysteinewere obtained.

In this case, the mutant obtained from the agar culture medium to whichonly S-aminoethyl-L-cysteine was added, was the 81512 strain. The mutantobtained from the agar culture medium to which S-aminoethyl-L-cysteineand glycine were added, was the 11011A-1 strain (deposit No. 1109 ofmicroorganisms of FRI). The mutant obtained from the agar culture mediumto which S-aminoethyl-L-cysteine and L-threonine were added, was the81502 strain.

Production of εPL:

S-Aminoethyl-L-cysteine tolerant 81512 strain in quantities of aplatinum earpick was inoculated in 5 ml of the culture medium having thesame composition as that of the above first culture medium, cultivatedwith shaking at 30° C. for eight days. At the conclusion of thecultivation, the concentration of εPL in the culture solution wasdetermined by the method of Itzhaki.

The yield of εPL per liter of the culture solution was 0.67 gr.

EXAMPLES 2 and 3

Using the same method as used in Example 1 except that the mutantsstrain 81512 strain tolerant to S-aminoethyl-L-cysteine was changed tothe mutant strain 11011A-1 tolerant to S-aminoethyl-L-cysteine andglycine (deposit No. 1109 of microorganisms of FRI) (Example 2) and themutant 81502 strain tolerant to S-aminoethyl-L-cysteine and L-threonine(Example 3), εPL was produced and its concentration was determined bythe same method as in Example 1.

The yields of εPL per liter of the culture solution were 0.88gr.(Example 2) and 0.72 gr.(Example 3), respectively.

EXAMPLE 4 Obtention of the chloramphenicol-treated mutant strains:

The mutant strain tolerant to S-aminoethyl-L-cysteine that was obtainedin Example 1 was inoculated in 5 ml of the culture medium having thesame composition as that of the first culture medium as described inExample 1.

After shaking the culture medium at 30° C. for two days, chloramphenicolin quantities from 50 to 500 mg per liter of the culture solution,preferably 100 mg, was added to the solution, and the cultivation wascontinued for further 5-10 hours, preferably eight hours. The cultivatedbacteria were collected by centrifugation, washed with sterilized wateror a physiological saline solution and applied to an agar culture mediumhaving the same composition as that of the first culture medium inaddition to 1.7% of agar.

After static cultivation at 30° C. for 8 days, a conventional agarculture medium containing Staphylococcus aureus was overlapped on thesaid medium. After cultivating further one night, the strains whichformed a large zone of inhibition of growth of Staphylococcus aureuswere the desired chloramphenicol-treated mutant 50833 stain deposit No.1110 of microorganisms of FRI).

Production of εPL:

Using the same method as described in Example 1 except that the 81512strain was changed to the obtained plasmid amplified 50833 strain, εPLwas produced and its concentration was determined by the same method asin Example 1.

The yield of εPL per liter of the culture solution was 1.80 gr.

COMPARATIVE EXAMPLE 1

Using the same method as described in Example 1 except that strain ofthe mutant 81512 strain tolerant to S-aminoethyl-L-cysteine was changedto Streptomyces albulus subsp. lysinopolymerus No. 346-D strain, εPL wasproduced and its concentration was determined by the same method as inExample 1.

The yield of εPL per liter of the culture solution was 0.20 gr. clEXAMPLE 5

Using the same method as described in Example 4 except that theproduction of εPL was conducted as follows.

The chloramphenicol-treated mutant 50833 in quantities of a platinumearpick were inoculated in 5 ml of the culture medium having the samecomposition as that of the above culture medium with the addition to0.5% of L-lysine monohydrochloride, cultivated with shaking at 30° C.for eight days. At the conclusion of the cultivation, the concentrationof εPL in the culture solution was determined by the method of Itzhaki.The yield of εPL per liter of the culture solution was 1.85 gr.

COMPARISON EXAMPLE 2

Using the same method as described in Example 5 except that thechloramphenicol-treated mutant 50833 strain was changed to Streptomycesalbulus subsp. lysinopolymerus No. 346-D strain, εPL was produced andits concentration was determined by the same method as in Example 5.

The yield of εPL per liter of the culture solution was 0.16 gr.

EXAMPLE 6

To 1.5 liter of the culture medium containing the same component as thatof the first culture medium described in Example 1, 0.05 volume % of adefoaming agent of a polyoxyalkylene glycol derivative was added. Theculture solution 50 ml in which the mutant 11011A-1 strain tolerant toS-aminoethyl-L-cysteine was precultivated was inoculated in the saidmedium, and the mutant was cultivated with shaking of 600 rpm at 30° C.in an air flow rate of 2 1/min..

After 24 hours, 5% of glucose and 1% of ammonium sulfate were sterilelyadded to the medium. After lowering of the pH, 6N of sodium hydroxidewas added while the pH was automatically and continuously controlledwith a pH controller so as not to ball below 4.0. After the cultivation,the bacteria were removed with a centrifuge and εPL in the culturesolution was purified with an anion exchange resin of IRA-402, a cationexchange resin of IRC-50 and active carbon of Carboraffin 50 w, and thepurity of the obtained εPL was 99.9 weight % and the yield of εPL perliter of the culture solution was 4.77 gr.

COMPARATIVE EXAMPLE 3

Using the same method as described in Example 6 except that the mutant11011A-1 strain tolerant to S-aminoethyl-L-cysteine was changed toStreptomyces albulus subsp. lysinopolymerus No. 346-D strain, εPL wasproduced. The purity of the obtained εPL was 97.8 weight % and the yieldof εPL per liter of the culture solution was 0.56 gr.

EXAMPLE 7

To 1.5 liter of the culture medium containing the same component as thatof the first culture medium described in Example 1, 0.05 volume % of adefoaming agent of a polyoxyalkylene glycol derivative was added. 50 mlof the culture solution in which the chloramphenical-amplified treatedmutant 50833 strain was precultivated was inoculated in the said medium,and the mutant was cultivated with aeration and stirring at 30° C. foreight days. When the pH of the culture solution began to drop, 2.5% ofglucose and 0.5% of L-lysine monohydrochloride were sterilely added tothe medium. Thereafter, so as not to lower the glucose concentration ofthe culture solution below 2%, 2.5% of glucose was sterilily addedsuccessively. After lowering the pH, 6N of sodium hydroxide was addedwhile the pH was automatically and continuously controlled with a pHcontroller so as not to decrease below 4.0.

After the cultivation, the bacteria were removed with a centrifuge andthe concentration of εPL in the culture solution was determined by themethod Itzhaki. The yield of εPL per liter of the culture solution was20.3 gr.

COMPARISON EXAMPLE 4

Using the same method as described in Example 7 except that thechloramphenical-treated mutant 50833 strain was changed to Streptomycesalbulus subsp. lysinopolymerus No. 346-D strain, the concentration ofεPL was determined by the same method as in Example 7.

The yield of εPL per liter of the culture solution was 0.20 gr.

EXAMPLE 8

To 1.5 liter of the culture medium containing the same component as thatof the first culture medium described in Example 1, 0.05 volume % of adefoaming agent of a polyoxyalkylene glycol derivative was added. 50 mlof the culture solution in which the chloramphenical treated 50833strain was precultivated was inoculated in the said medium, and themutant was cultivated with shaking of 600 rpm at 30° C. in an air flowrate of 2 1/min..

After 24 hours, as the pH of the culture solution began to decrease, aglucose solution and a L-lysine solution were passed through the culturetank so as to maintain the concentrations of 1% of gulucose and 0.2% ofL-lysine, and exhausted the culture solution. After the pH was lowered,6N of sodium hydroxide was added while the pH was automatically andcontinuously controlled with a pH controller so as not to decrease below4.0.

After the cultivation, the bacteria were removed with a centrifuge, andεPL in the culture solution was purified with an anion exchange resin ofIRA-402, a cation exchange resin of IRC-50 and active carbon ofCarboraffin 50w. Then εPL was crystallized with alcohol. The purity ofthe obtained εPL was 99.9 weight % and the yield was 5.02 gr.

We claim:
 1. A method for producing epsilon poly-L-lysinecomprising:culturing Streptomyces albulus subsp. lysinopolymerus strainFERM-BP-1109 in a culture medium containing L-lysine, said culturemedium further containing a source of carbon, a source of nitrogen,inorganic salts and vitamins to produce epsilon-poly-L-lysine; andrecovering the produced epsilon-poly-L-lysine from the culture.
 2. Themethod according to claim 1 wherein said source of carbon includes atleast one sugar.